Here we develop a microdroplet sensor based on bipolar electrochemistry and electrochemiluminescence (ECL) imaging. The sensor was constructed with a closed bipolar cell on a hybrid poly(dimethylsioxane) (PDMS)-indium tin oxide (ITO) glass microchip. The ITO microband functions as the bipolar electrode and its two poles are placed in two spatially separate micro-reservoirs predrilled on the PDMS cover. After loading microliter-sized liquid droplets of tris(2,2'-bipyridyl) ruthenium (II)/2-(dibutylamino) ethanol (Ru(bpy)3(2+)/DBAE) and the analyte to the micro-reservoirs, an appropriate external voltage imposed on the driving electrodes could induce the oxidation of Ru(bpy)3(2+)/DBAE and simultaneous reduction of the analyte at the anodic and cathodic poles, respectively. ECL images generated by Ru(bpy)3(2+)/DBAE oxidation at the anodic pole and the electrical current flowing through the bipolar electrode can be recorded for quantitative analyte detection. Several types of quinones were selected as model analytes to demonstrate the sensor performance. Furthermore, the cathodic pole of bipolar electrode can be modified with (3-aminopropyl)triethoxysilane-gold nanoparticles-horseradish peroxidase composites for hydrogen peroxide detection. This microdroplet sensor with a closed bipolar cell can avoid the interference and cross-contamination between analyte solutions and ECL reporting reagents. It is also well adapted for chemical analysis in the incompatible system, e.g., detection of organic compounds insoluble in water by aqueous ECL generation. Moreover, this microdroplet sensor has advantages of simple structure, high sensitivity, fast response and wide dynamic response, providing great promise for chemical and biological analysis.